Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis.

BackgroundKey effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC).MethodsWe used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells.ResultsImmunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.ConclusionsOur data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer

Organosilica nanoparticles containing sodium borocaptate (BSH) provide new prospects for boron neutron capture therapy (BNCT): efficient cellular uptake and enhanced BNCT efficacy

Boron neutron capture therapy (BNCT), a method based on the fission of boron-10 upon neutron irradiation, has emerged as an attractive option for radiation therapy. To date, the main drugs used in BNCT are 4-boronophenylalanine (BPA) and sodium borocaptate (BSH). While BPA has been extensively tested in clinical trials, the use of BSH has been limited, mainly due to its poor cellular uptake. Here, we describe a novel type of mesoporous silica-based nanoparticle containing BSH covalently attached to a nanocarrier. Synthesis and characterization of these nanoparticles (BSH-BPMO) are presented. The synthetic strategy involves a click thiol–ene reaction with the boron cluster, providing hydrolytically stable linkage with the BSH in four steps. The BSH-BPMO nanoparticles were efficiently taken up into cancer cells and accumulated in the perinuclear region. Inductively coupled plasma (ICP) measurements of boron uptake in cells highlight the important role of the nanocarrier in the enhancement of boron internalization. BSH-BPMO nanoparticles were also taken up and distributed throughout tumour spheroids. BNCT efficacy was examined by the neutron exposure of the tumour spheroids. BSH-BPMO loaded spheroids were completely destroyed upon neutron irradiation. In contrast, neutron irradiation of tumour spheroids loaded with BSH or BPA resulted in significantly less spheroid shrinkage. The significant difference in BNCT efficacy of the BSH-BPMO was correlated with the improved boron uptake via the nanocarrier. Overall, these results demonstrate the critical role of the nanocarrier in BSH internalization and the enhanced BNCT efficacy of the BSH-BPMO compared with BSH and BPA, two drugs used in BNCT clinical trials

Designing Mesoporous Silica Nanoparticles to Overcome Biological Barriers by Incorporating Targeting and Endosomal Escape.

The several biological barriers that nanoparticles might encounter when administered to a patient constitute the major bottleneck of nanoparticle7 mediated tumor drug delivery, preventing their successful translation into the clinic and reducing their therapeutic profile. In this work, mesoporous silica nanoparticles have been employed as a platform to engineer a versatile nanomedicine able to address such barriers, achieving (a) excessive premature drug release control, (b) accumulation in tumor tissues, (c) selective internalization in tumoral cells, and (d) endosomal escape. The nanoparticles have been decorated with a self-immolative redox-responsive linker to prevent excessive premature release, to which a versatile and polyvalent peptide that is able to recognize tumoral cells and induce the delivery of the nanoparticles to the cytoplasm via endosomal escape has been grafted. The excellent biological performance of the carrier has been demonstrated using 2D and 3D in vitro cell cultures and a tumor-bearing chicken embryo model, demonstrating in all cases high biocompatibility and cytotoxic effect, efficient endosomal escape and tumor penetration, and accumulation in tumors grown on the chorioallantoic membrane of chicken embryos

Iodine containing porous organosilica nanoparticles trigger tumor spheroids destruction upon monochromatic X-ray irradiation: DNA breaks and K-edge energy X-ray

アインシュタインの光電効果をがん細胞の中で再現　放射線治療への新展開. 京都大学プレスリリース. 2021-07-14.Quantum physics helps destroy cancer cells. 京都大学プレスリリース. 2021-07-14.X-ray irradiation of high Z elements causes photoelectric effects that include the release of Auger electrons that can induce localized DNA breaks. We have previously established a tumor spheroid-based assay that used gadolinium containing mesoporous silica nanoparticles and synchrotron-generated monochromatic X-rays. In this work, we focused on iodine and synthesized iodine-containing porous organosilica (IPO) nanoparticles. IPO were loaded onto tumor spheroids and the spheroids were irradiated with 33.2 keV monochromatic X-ray. After incubation in CO₂ incubator, destruction of tumor spheroids was observed which was accompanied by apoptosis induction, as determined by the TUNEL assay. By employing the γH2AX assay, we detected double strand DNA cleavages immediately after the irradiation. These results suggest that IPO first generate double strand DNA breaks upon X-ray irradiation followed by apoptosis induction of cancer cells. Use of three different monochromatic X-rays having energy levels of 33.0, 33.2 and 33.4 keV as well as X-rays with 0.1 keV energy intervals showed that the optimum effect of all three events (spheroid destruction, apoptosis induction and generation of double strand DNA breaks) occurred with a 33.2 keV monochromatic X-ray. These results uncover the preferential effect of K-edge energy X-ray for tumor spheroid destruction mediated by iodine containing nanoparticles

Systematic Identification of Cellular Signals Reactivating Kaposi Sarcoma–Associated Herpesvirus

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma–associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma–derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets